Cryptococcus neoformans is an opportunistic pathogen, responsible for life-threatening disease in individuals who are immunocompromised due to AIDS or other conditions. Current therapy for cryptococcosis is inadequate. Fungal glycan structures offer appealing potential targets for specific chemotherapy, because they differ substantially from those found in host organisms. Carbohydrate structures in C. neoformans modify proteins and lipids, compose most of the cell wall, and form an elaborate polysaccharide capsule that surrounds the cell wall and is required for virulence. Notably, the capsule and cell wall are critical structures that are completely absent from mammalian cells, and the cell wall has already been validated as a drug target. We have discovered a novel glycosylating enzyme in C. neoformans, which transfers xylose-phosphate from UDP-xylose to mannose residues. This activity is unprecedented in nature. In this R21 application, we propose to pursue the hypothesis that this enzyme is important in cryptococcal biology, and to explore its biochemical activity with an eye to novel function and the development of screening assays. As proposed in Aim I, biochemical studies of the protein will address the reactants and kinetics of the observed activity, as well as critical residues and improved assays. The cellular context of the enzyme will be determined by studies of localization and potential binding partners in Aim II. Finally, the role of this unique protein in biology will be addressed using cells genetically engineered to lack the activity. These will be compared to parental strains to assess alterations in glycosylation and related phenotypes in Aim III. Methods will include biochemical assays, glycan analysis, epitope-tagging, imaging, metabolic radiolabeling, and peptide analysis. This multi-faceted strategy will elucidate the activity of a unique protein and provide fundamental understanding of fungal biology and glycan synthesis. PUBLIC HEALTH RELEVANCE: This research is highly relevant to public health because the organism being investigated causes serious human illness, for which current therapies are not adequate. Further, this work will contribute to basic science knowledge, which will impact the study of pathogenic microbes and other areas of biology.